We are currently characterizing the abnormalities for regulation of human muscle glycogen synthase in insulin-resistant subjects. In insulin-resistant subjects, fasting glycogen synthase phosphatase activity is reduced and fails to show the peak insulin stimulation observed for insulin-sensitive subjects at 10 minutes. Similar results were obtained using phosphorylase as substrate for the phosphatase activity with the exception that insulin-sensitive subjects maintained the stimulated level of activity from 20 minutes until the end of the insulin infusion. Using specific inhibitors the abnormal enzyme activity was identified as a type-1 phosphatase in human muscle from insulin-resistant subjects. The reduced type-1 activity observed in both glycogen and cytosolic subcellular fractions suggested an alteration in the catalytic subunit independent of the glycogen-bound regulatory subunit or G component. The abnormally low fasting type-1 phosphatase activity in insulin resistant subjects persisted following trypsin treatment, suggesting that inhibitors 1 and 2 (characterized regulators of type-1 phosphatase) are not important, determinants of the abnormal phosphatase activity. Western blots indicate an increased concentration of catalytic subunit for type-1 phosphatase in the muscle from insulin-resistant subjects. This result suggests that the intrinsic activity or regulation of the catalytic subunit is abnormal in insulin-resistant subjects. The apparent affinity for cAMP activation of muscle cAMP-dependent protein kinase (CPK) is not decreased in insulin-resistant subjects following insulin infusion. In contrast, sensitive subjects show a reduced apparent affinity of their CPK regulatory subunit for cAMP following insulin infusion. This results in inactivation of the kinase and could stimulate glycogen synthase activity. These results suggest that abnormal regulation of both glycogen synthase phosphatase and CPK contribute to the insulin resistance which is secondary to abnormal insulin-stimulated glycogen synthase activity.